2 n h2so4

High binding extra 96-well plates (Immulon 4; Dynatech Laboratories, Chantilly, VA) were coated with 100 μL of human-specific goat anti-AAT (1:2,000 diluted; Bethyl Laboratories, Montgomery, TX) in Voller’s buffer overnight at 4°C. After blocking with 1% nonfat dry milk in PBS with Tween 20 (PBST), duplicate standard curves (Athens Research and Technology, Athens, GA) and serially diluted experimental samples were incubated in the plate at RT for 1 h, and a second goat anti-hAAT (horseradish peroxidase) antibody (1:5,000 dilution; Bethyl Laboratories) was incubated at RT for 1 h. The plate was washed with PBST between reactions. After reaction with 3,3′, 5,5′ tetramethylbenzidine dihydrochloride peroxidase substrate (KPL, Gaithersburg, MD), reactions were stopped by adding 2 N H₂SO₄ (Fisher Scientific, Hudson, NH). Plates were read at 450 nm on a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA). The cell culture, transfection, and ELISA studies were performed as previously described.¹⁶ (link)

An enzyme-linked immunosorbent assay (ELISA) was used for the detection of anti-Ag85B antibodies in the sera of the immunized animals. Briefly, the purified protein derivative (PPD) or the Ag85B protein was adjusted to 2 μg/mL with coating buffer (0.05 M Na₂CO₃-NaHCO₃, pH 9.6), and 100 µL was added to each well of 96-well Immunosorp plates (Nunc, Weston, FL, USA) for incubation overnight at 4 °C. The plates were washed three times with 375 μL PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, and 0.05% Tween-20) and then blocked in 0.2 mL 3% bovine serum albumin in PBS for 2 h at room temperature. After three washes, the collected serum samples (diluted 1:200 in blocking buffer) were added to each well and incubated at 37 °C for 1 h. After five washes five times, 100 µL of 1:5000-diluted HRP-conjugated anti-human IgG, IgG1, or IgG2 secondary antibody (Promega, Madison, WI, USA) was added for incubation at 37 °C for a further 1 h. After five washes, the color reaction was developed with 100 µL TMB (3, 3′, 5, 5′-tetramethylbenzidine) substrate solution (eBioscience, San Diego, CA, USA) and stopped via the addition of 50 µL 2 N H2SO4 (eBioscience, San Diego, CA, USA). The optical density (OD) was measured and recorded at a wavelength of 450 nm using a microplate reader (ELX50, Bio-Tek Instruments, Salem, MA, USA).